Aspects of recombination in RNA viruses IntroductionRNA viruses deserve their reputation as Nature's swiftest evolvers. Their high rates of mutation and replication allow them to move through sequence space at a pace that often makes their DNA-based hosts ' evolution lookglacial by comparison. Over the last two decades it has become increasingly clear that many RNA viruses add the capacity to exchange genetic material with one another, and to acquiregenes from their hosts, to this evolutionary repertoire. So, in addition to producing prodigious amounts of the raw material of evolution (mutations), these viruses also possess mechanisms that, in principle, allow them both to purge theirgenomes of accumulated deleterious changes (Muller, 1964) and to create or spread bene locial combinations of mutations in an efficient manner (Fisher, 1930;Muller, 1932), two processes which are not available to clonal organisms. Two distinct but not mutually exclusive types of genetic exchange operate in RNA viruses. The first, reassortment,occurs only in multipartite viruses and involves swapping oneor more of the discrete RNA molecules that make up the segmented viral genome. Antigenic shift in in¯uenza A virus is an example of this sort of genetic exchange and serves as a good illustration of the potential evolutionary significance ofsuch events. A second process, recombination, can occur ineither segmented or unsegmented viruses when `donor'nucleotide sequence is introduced into a single, contiguous` acceptor ' RNA molecule to produce a new RNA containinggenetic information from more than one source. In this paperwe focus on this type of genetic exchange. First, we brie¯yreview current knowledge of RNA virus recombination anddescribe new methods for detecting its occurrence using genesequence data. We then discuss some of the evolutionaryimplications of virus recombination and some of the constraintsthat may shape the variety of RNA virus recombination.

Recombination in RNA virusesIn some cases of RNA virus recombination, the donorsequence neatly replaces a homologous region of the acceptorsequence leaving its structure unchanged. This has beenclassified as `homologous recombination ' (Lai, 1992) since itinvolves not just homologous parental RNAs, but alsocrossovers at homologous sites. However, this is not alwaysthe case ; hybrid sequences resulting from aberrant homologousrecombination (when similar viruses exchange sequence withoutmaintaining strict alignment) and nonhomologous recombination(recombination between unrelated RNA sequences)are also commonly observed (Lai, 1992).Despite producing distinct kinds of hybrid RNAs, as well asdefective interfering (DI) RNAs (Lazzarini et al., 1981), thedifferent types of recombination appear to be variations on acommon theme. To date, almost all studies on the mechanismsof recombination in RNA viruses have supported a copy±choice model, originally proposed in the case of poliovirus(Cooper et al., 1974) and now well studied in a number ofexperimental systems (Duggal et al., 1997; Jarvis & Kirkegaard,1992; Kirkegaard & Baltimore, 1986; Nagy & Bujarski, 1995,1998; Nagy et al., 1998; Simon & Nagy, 1996; for a recentreview see Nagy & Simon, 1997). Under this model, hybridRNAs are formed when the viral RNA-dependent RNApolymerase complex switches, mid-replication, from one RNAmolecule to another. This results in homologous recombinationif the replicase continues to copy the new strand preciselywhere it left the old one, and aberrant or nonhomologousrecombination if it does not. This template-switching mechanismis fundamentally different from the enzyme-drivenbreakage±rejoining mechanism of homologous recombinationin DNA, not least because it invokes replication as a necessarycomponent of the process. Finally, Chetverin et al. (1997)presented evidence for a splicing-like, transesterication mechanismto explain the in vitro generation of recombinantsbetween RNAs associated with Qb bacteriophage ± a possibleexception to the copy±choice model of recombination in RNAviruses. Whether such a mechanism operates in vivo remains tobe seen ; however, end-to-end joining is not regarded as alikely mechanism for homologous recombination.There is now a fairly rich literature documenting recombinationin RNA viruses. Many excellent recent reviewshave dealt comprehensively with aspects of recombination inexperimental and natural settings with respect to animalviruses (Lai, 1992, 1996; Ball, 1997; Strauss & Strauss, 1997),plant viruses (Lai, 1992; Simon & Bujarski, 1994; Roossinck,1997; Aaziz & Tepfer, 1999) and bacteriophages (Mindich,1996; Chetverin, 1997). Recently, reports describing homologous recombination in rotaviruses (Suzuki et al., 1998) and in hantaviruses (Sibold et al., 1999) have added doublestrandedand negative-sense RNA viruses, respectively, to thelong list of RNA viruses in which homologous recombinationhas been detected. The emerging signi®cance of RNA virusrecombination is all the more fascinating given the fact that ±until the comparatively recent publication by Cooper et al.(1974) which showed that mutants of poliovirus could bemapped by recombination analysis ± recombination was notthought to be a property of RNA genomes.

New tools for detecting recombination invirusesThe molecular revolution initiated by the development ofPCR has transformed the study of virus recombination.Sequence analysis and phylogenetic techniques have in recentyears proven to be extremely effective methods for detectingand characterizing recombination events among RNA virusesboth in nature (Gao et al., 1998; Hahn et al., 1988; Holmes etal., 1999; Kusters et al., 1990; Revers et al., 1996; Sibold et al.,1999; Suzuki et al., 1998; Worobey et al., 1999) and in thelaboratory (Banner&Lai, 1991; Greene&Allison, 1994; Kotieret al., 1995; Mindich, 1996; Palasingam & Shaklee, 1992;Weiss & Schlesinger, 1991). They offer a way not just torecover information about recombination events that mayhave occurred long ago or are exceedingly rare (Snijder et al.,1991; Weaver et al., 1997), but also to probe the nest detailsof the mechanism itself (Banner & Lai, 1991; Olsthoorn & vanDuin, 1996). They also provide a means to home in on theprecise location of putative crossover points and to test resultssuggestive of recombination for statistical signicance.Several methods for detecting recombination events andlocating breakpoints are graphical in nature, exploiting the factthat many recombinant sequences are mosaics comprisingregions with quite different phylogenetic histories. One ofthese, Split Decomposition analysis (Bandelt & Dress,1992; Huson, 1998), presents con¯icting phylogenetic signalin a single diagram. If no recombination has occurred in thesequences tested, the splits-graph tends to resemble adichotomously branching phylogenetic tree, because thisadequately describes sequence relationships. However, indatasets containing con¯icting signal due to the presence ofrecombinant and hence `mosaic' sequences, the tree-likepattern is often replaced by a more complicated `network' thatindicates a history of genetic exchange. It is worth noting thatconventional phylogenetics programs are constrained toproduce simple branching trees and can lead to seriousmisinterpretation if sequence alignments are not carefullyexamined for evidence of recombination prior to tree reconstruction.Several other graphical applications, including ` bootscanning'(Salminen et al., 1995), `PhylPro' (Weiller, 1998),`TOPAL' (McGuire &Wright, 1998) and `DIVERT' (Gao et al.,1998), use ` sliding windows' to detect discordant sequencerelationships suggestive of recombination. Bootscanning is anaptly named phylogenetic approach that initially produces atree from a small window at one end of a sequence alignmentand assesses its robustness using bootstrapping. The windowis then incrementally shifted along the alignment and a newbootstrap tree is produced for each resulting subset of thealignment. Significant topological changes in the position of asequence in different windows indicate possible recombination.PhylPro and TOPAL both slide a pair of adjacent windowsalong the sequence alignment. Each of these methods employsa different measure of phylogenetic signal, but in both thephylogenetic information contained in one window is comparedto that in the neighbouring window. In the absence ofrecombination all windows are expected to show similarpatterns. On the other hand, if recombination has occurred,some adjacent windows are expected to contain con¯ictingsignal and the difference between them should be greatestwhen they straddle a recombination breakpoint. DIVERT, thesimplest of the sliding window graphical methods (and oftenthe most effective), outputs a graph of genetic distancecomparisons between a chosen sequence and comparisonsequences, which can show runs of sequence similarity anddissimilarity suggestive of recombination. Diversity plots havebeen used to great effect in the search for recombinant humanimmunodeficiency virus and simian immunodeficiency virusstrains (Gao et al., 1998, 1999) and are ideally suited fordetection of RNA virus homologous recombination (Worobeyet al., 1999).

Many of these programs permit a simple qualitativeassessment of possible recombination breakpoints based on thevisual analysis of their output. However, for cases whereputative recombinants and reasonably close relatives of theiracceptor and donor sequences are available, more sophisticatedprocedures exist for locating crossover points. InformativeSites Analysis (Robertson et al., 1995), a parsimony-basedadaptation of the maximum test (Maynard Smith, 1992),uses the distribution of polymorphic sites between a probablerecombinant and its putative ` parents ' to estimate recombinationjunctions. The results can then be compared torandomized distributions of polymorphic sites to assess theirsigni®cance. A similar method, LARD (Holmes et al., 1999),uses a maximum likelihood method to infer the optimalbreakpoints for a possible recombinant, then uses simulatedsequences to test the statistical significance of the results.Some other methods attempt to quantify the amount ofrecombination between a set of sequences, rather thandocument specific recombination events, often using thedegree of linkage equilibrium. One way this can be done iswith the Index of Association. Using this statistical test, whichwas designed to detect associations between alleles at differentloci, it is possible to measure the extent of linkage equilibriumwithin populations (Maynard Smith et al., 1993). Another,based on a direct phylogenetic analysis, is the Homoplasy Test

Here, the number ofhomoplasic (i.e. convergent and parallel) base changes in dataobserved after construction of a maximum parsimony tree iscompared to that number expected by chance. Excessivehomoplasies are the fingerprint of recombination.

How clonal are viruses?The impressive tally of recombinant ` hopeful monsters' ±dramatically altered hybrid viruses that have passed the test ofnatural selection and emerged as successful new viruses ±attests to the power of recombination as an evolutionary forcein RNA viruses (Allison et al., 1989; Gibbs & Cooper,1995; Herrewegh et al., 1998; Luytjes et al., 1988; Revers et al.,1996; Ro$hm et al., 1996; Snijder et al., 1991; Suzuki et al.,1998; Weaver et al., 1997). In some cases, evolutionaryevidence suggests that newly formed recombinant strains mayhave initially exhibited decreased functionality, but subsequentlyevolved to compensate for these effects. Forexample, the Western equine encephalitis (WEE) complexviruses are the product of a single recombination event thatoccurred between Eastern equine encephalitis virus (EEEV) anda Sindbis-like virus, probably within the last 2000 years(Weaver et al., 1997). Sequence analysis of WEEV indicatedthat, subsequent to the recombination event, its EEEV-likecapsid protein evolved to become more like a Sindbis-viruscapsid, possibly because it needs to interact with Sindbis-likeglycoproteins during virus budding (Hahn et al., 1988).Conversely, in a sort of evolutionary compromise, almost allthe amino acid changes in the Sindbis-like glycoproteins havebeen to residues that match those of EEEV. It is possible thatthe new antigenic properties conferred by the Sindbis-likeglycoproteins of this predominantly EEEV-like hybrid weresufficiently advantageous to off-set what appears to have beena signi®cant mismatch between its recombinant structuralproteins.Whatever the circumstances of the survival and subsequentdiversi®cation of particular recombinants, it is now evidentthat many pass through the narrow gates of natural selectionand contribute to the diversity seen in RNA viruses. Theproduction of new strains having genomes comprising regionswith different histories has important implications for the waywe think about virus evolution. For one, it means that there isno single phylogenetic tree that can describe the evolutionaryrelationships between viruses ; the recombinative nature ofviruses simply precludes the possibility of a ` true ' phylogenetictaxonomy. Far from being an incidental process best ignoredwhen considering virus relationships and history, recombinationmay have played a crucial role in generating many ofthe taxonomic groups we recognize (Koonin & Dolja, 1993) ±today's hopeful monster giving rise to tomorrow's genus orfamily of viruses. Some studies of recombination have even ledto recommendations that higher-than-family taxonomic unitsshould be avoided altogether (Goldbach, 1992).The inappropriateness of tree-like representations of evolutionaryrelatedness may also apply within some virus species.In principle, the possibility of homologous recombinationbetween similar strains means that the population structure inviruses could range from completely clonal, if no recombinationhas taken place, to panmictic, if recombination hasbeen common enough to effectively randomize loci. Althoughsigni®cant progress has been made studying populationstructure in other organisms (Maynard Smith et al., 1993),similar work remains to be done for RNA viruses.Many taxonomic groupings of viruses ± for example boththe Togaviridae and the Coronaviridae ± include members that,while clearly sharing homologous genes, differ in the order inwhich these genes are organized along the genome, anotherindication that recombination has been important in theirevolutionary history. Other cases ± exemplied by the haemagglutinin-esterase gene known to be present in at least threevirus genera (Snijder et al., 1991) ± show recombination as theengine driving modular evolution, whereby functional modulesfrom different sources are brought together to create newviruses. The discovery of recombination in an increasingnumber of viruses, in addition to presenting phylogenetic andtaxonomic difficulties, challenges the desirability of using shortsequence regions as markers for entire virus genomes (forinstance in molecular epidemiology studies) since they maynot accurately re¯ect true genetic or antigenic characteristics.

Evolutionary advantages of recombinationTheoretical explanations for the evolution of recombinationand hence some aspects of sexual reproduction tend to ®t intoone of two standard classes : (1) that it enables the creation andspread of advantageous traits, and (2) that it permits theremoval of deleterious genes (for a review see Hurst & Peck,1996). This second explanation is often linked to the notion of`Muller's ratchet ' ± the random loss of those individuals in apopulation having the fewest deleterious alleles (Muller, 1964).Muller's ratchet predicts the gradual build-up of deleteriousalleles, asexual population. Experiments with RNAviruses, one of only a handful of organisms in which thishypothesis has actually been put to the test, have generallysupported its operation and shown decreased ®tness forpopulations in which it occurs (Chao et al., 1992). Experimentalevidence (Chao et al., 1997) also shows that sex (in this casereassortment) can reduce the mutational load in a populationand so help it escape from accumulated deleterious effects.Although such direct experimental evidence has yet todemonstrate a similar advantage for recombination, in principleit too could serve to efficiently remove disadvantageous allelesfrom a population by combining mutation-free parts ofdifferent genomes. Indeed, suggestions have been made thatreassortment in segmented RNA viruses and recombination inmonopartite RNA viruses represent alternative evolutionarystrategies for genetic exchange in this group (Chao et al., 1992).While this idea is fascinating, it is interesting to note thatreassortment and recombination are not mutually exclusiveand that several segmented viruses also experience recombination,sometimes frequently. These include the bacteriophageu6 (Mindich et al., 1992), rotaviruses (Suzuki et al., 1998),in¯uenza A virus (Khatchikian et al., 1989), hantaviruses (Siboldet al., 1999), ¯ock house virus (Li & Ball, 1993) and many plantviruses (Bujarski & Kaesberg, 1986; Greene & Allison, 1994;Robinson, 1994; Rott et al., 1991). As if to prove this point,Masuta et al. (1998) recently reported an interspeci®c hybrid oftwo cucomoviruses that arose by both reassortment andrecombination.Nonetheless, a great deal of evidence indicates that someRNA viruses do benefit from the genome-purging effects ofrecombination. A multitude of experimental studies haveshown that weak or even non-replicative mutant strains canrecombine to form viable, highly viruses. Examples includethe functional chimeras formed between nonreplicating RNAsand DI RNAs of tombusviruses (White & Morris, 1994),infectious recombinants produced by different combinationsof mutationally altered Sindbis virus RNAs (Raju et al.,1995; Weiss & Schlesinger, 1991) and wild-type revertantrecombinants of Qb phage mutants (Palasingam & Shaklee,1992) and of bromovirus mutants (Rao & Hall, 1993).Plant viruses have also been observed to repair theirgenomes by recombining with host transgene transcripts(Borja et al., 1999; Gal-On et al., 1998; Greene & Allison,1994; Rubio et al., 1999). Similarly, in one experiment with adeletion mutant of mouse hepatitis virus (MHV) transfectedwith a synthetic RNA that contained the deleted region(Koetzner et al., 1992), and another with an in¯uenza A virusmutant with a damaged neuraminidase gene (Bergmann et al.,1992), recombination successfully repaired defective genes.Studies of recombination in bacteriophages, too, indicate arepair function for recombination (Mindich et al., 1994). RNArecombination even appears to provide a telomerase-likefunction by repairing the 3« ends of satellite RNAs of bothturnip crinkle virus (Burgyan & Garcõ!a-Arenal, 1998) andcucumber mosaic virus (Simon & Nagy, 1996).Unintentional ` natural experiments' with some virusespoint to the same conclusion. The frequent recovery ofrecombinant isolates of poliovirus (Georgescu et al., 1994; Kew& Nottay, 1984) and infectious bronchitis virus (Jia et al.,1995; Kusters et al., 1990; Wang et al., 1994) that result fromrecombination involving vaccine strains shows that recombinationhas the potential to produce ` escape mutants' innature as well as in experiments. Recently, recombination hasalso been detected in other RNA viruses for which multivalentvaccines are in use or in trials (Holmes et al., 1999; Suzuki et al.,1998; Worobey et al., 1999). We think the potential forrecombination to produce new pathogenic hybrid strains, andthe possible impact of such escape recombination, needs to becarefully considered whenever multivalent live-attenuatedvaccines are used to control RNA viruses. Assumptions thatrecombination either does not happen or is unimportant inRNA viruses have a history of being proved wrong.In addition to the evidence favouring a role for geneticexchange in eliminating deleterious alleles, many recombinantRNA virus strains provide ample indication that recombinationcan generate beneficial new variation. In some viruses this newvariation is achieved by borrowing genetic material from theirhosts. One intriguing example of this is bovine viral diarrhoeavirus (BVDV), a pestivirus that recombines with host cellularprotein-coding RNA. As a result of virus±host recombinations,cytopathogenic BVDVs can develop from non-cytopathogenicones and cause a lethal syndrome, mucosal disease, in the hosts(Meyers et al., 1989). In¯uenza A virus has also been observedto recombine with cellular RNA, resulting in increasedpathogenicity for the hybrid viruses (Khatchikian et al., 1989).Recombination between virus and host genetic materialevidently occurs in plant viruses as well, as illustrated by aluteovirus isolate with 5«-terminal sequence derived from achloroplast exon (Mayo & Jolly, 1991) and closteroviruseswhich have acquired host cellular protein-coding genes (Doljaet al., 1994) which are nonessential for replication and virionproduction (Peremyslov et al., 1998).A link between recombination and increased pathogenicityhas also been revealed in cases that do not involve recombinationwith host genes. Template jumping duringreplication in viruses infecting cats has produced, on multipleoccasions, the pathogenic strains known as feline infectiousperitonitis viruses (FIPVs) by altering asymptomatic felineenteric coronaviruses, differing from them only by deletions ofaround 100 bp in predictable locations (Vennema et al., 1998).Another coronavirus, feline coronavirus (FCoV) type II,appears to be a homologous (or aberrant homologous)recombinant of FCoV type I and canine coronavirus(Herrewegh et al., 1995). Like FIPVs, FCoV type II viruses mayhave arisen on different occasions from separate recombinationevents (Motokawa et al., 1996).Experimental studies provide further signs of the ability ofrecombination to generate useful, new variation. In oneparticularly striking display of this, MS2 phage mutants lackingthe sequence for important stem-and-loop secondary structuresrepeatedly reconstructed them via nonhomologous recombination(Olsthoorn & van Duin, 1996).

Constraints on recombination

Recombination clearly plays a significant role in theevolution of RNA viruses by generating genetic variation, byreducing mutational load, and by producing new viruses. Weexpect that with current advances in sequencing and sequenceanalysis many more examples of hybrid viruses, produced byCFDI

recombination between different strains, will soon be found.However, it is clearly not the case that all RNA viruses areequally prone to recombination. It has still not been detected inseveral viruses despite strenuous searches (e.g. Bilsel et al.,1990), although some ± not otherwise known to recombine ±nevertheless produce DI RNAs. Amongst those known toproduce hybrids, the frequency of recombinants detected innatural or experimental studies varies markedly. Given thepotential advantages of recombination, why is there apparentlyso much variation between viruses in its occurrence ? While itis too soon to provide a de®nitive answer to this crucialquestion, it is possible to dissect the process that gives rise torecombinants and to consider the constraints that could act ateach stage to inhibit it. A simple model of recombinationbetween different RNA viruses, with possible constraints, ispresented in Fig. 1.In a sense, recombinogenic viruses are all alike in that theysuccessfully pass through each stage outlined in the model.Every non-recombining virus, on the other hand, is different inits own way since constraints that block recombination couldact by breaking any link in the chain and could involve not justviral genetic factors, but host and ecological factors particularto that virus. The first prerequisite for successful recombination(Fig. 1) is that an individual host must be infected by differentvirus strains. [This is not quite true, of course, since (1)recombination sometimes involves host RNA, (2) recombinationcould occur between viruses that have divergedwithin a clonally infected individual, and (3) evolutionarilyinvisible recombination could occur between identical RNAmolecules.] Host coinfection might never occur with someviruses simply because their divergent forms do not usuallyoverlap in space and time. Multivalent live-attenuated vaccinesCFDJ can be seen as potential risks in this context since they couldeffectively release some viruses from this constraint. Hostfactors could act at this stage too if, for instance, an immuneresponse reduces the window for simultaneous infection byquickly clearing a virus, or prevents superinfection altogetherby blocking secondary infections.Having successfully coinfected a single host, divergentviruses must next coinfect a single cell if recombination is toproceed. This step could be blocked by host factors, either byan immune response that keeps virus numbers low enough toprevent multiple infection of any individual cell, or by host cellgenetic factors that block entry of more than one virus particleinto a cell (Danis et al., 1993). Viral factors, interestingly, mightalso enforce significant constraints at this stage of the model.Recent evidence demonstrates that intracellular competitioncan be costly to viruses that infect the same host cell (Turner&Chao, 1998). Those that can keep cells to themselvesby limiting or preventing coinfection should be selectivelyfavoured, and many have evolved mechanisms to do just this(Simon et al., 1990; Singh et al., 1997; Turner et al., 1999). Oneof these, vesicular stomatitis virus (VSV), is an RNA virus inwhich recombination between different strains has not beendetected. Might superinfection exclusion be a constraint onrecombination in VSV? Another, the segmented bacteriophageu6, seems to limit excessive superinfection but not to theextreme of one-virus-per-cell that would preclude geneticexchange. Instead, it appears to have evolved an optimalcoinfection limit of two to three viruses per cell, presumably tobalance the costs of intracellular competition with the bene®tsof reassortment (Turner et al., 1999). Since the advantage (andcost) of recombination in any particular virus will be mediatedby such factors as the selective pressure for novel variation, theimportance of interactions between different parts of thegenome, as well as the virus mutation rate and population size,we should expect different optima (and therefore differentdegrees of constraint) in different cases.If divergent viruses manage to infect the same cell, the nextstep is simply for one of them to replicate in the presence of theRNA of the other. This is not necessarily inevitable even incoinfected cells. The replication of the u6 RNAs, for example,takes place within a procapsid and it is thought that the entryof two different RNA molecules of the same genomic segmentinto this sequestered environment is impossible or at least veryrare (Mindich et al., 1992). This could explain the lack ofhomologous recombination in this phage. Thus the vagaries ofRNA replication in certain viruses could impose physicalconstraints on the production of hybrids.Template switching by the viral replicase, the mechanismwhereby recombinant RNA molecules are actually created,may also be limited by physical constraints. The negativestrand RNA viruses,for example, whose genomes are packagedinto ribonucleoprotein structures by associationwith N protein, may be less permissive than other RNA virusesto copy±choice recombination. And perhaps the most importantphysical constraint on template switching ± particularlywith respect to homologous recombination ± is simplythe extent of sequence dissimilarity between potentiallyrecombining genomes. Finally, genetic variation in the susceptibilityof the viral replicase to jumping (Bujarski & Nagy,1996) no doubt plays a central role in determining how oftenand by what mechanism particular viruses recombine.Recombination occurs when these ®rst four steps areful®lled. Whether incipient recombinants persist, however,depends on the fifth and final step, the selective separation ofthe wheat from the chaff among hybrids. Although there isstrong evidence that genetic exchange can offer advantages insome circumstances, random recombination no doubt destroysmore good alleles than it creates. PCR studies which havemade possible the characterization of the initial products ofrecombination ± those present prior to removal by selection(Banner & Lai, 1991; Desport et al., 1998; Jarvis & Kirkegaard,1992) ± have produced important insights. Banner and Lai'sstudy of coronaviruses (1991), for example, showed that theinitial recombination events in their MHV system were almostentirely randomly distributed along the sequence investigated.It was only after passage through cell culture, with theopportunity for selection to remove less variants, thatcrossover sites became ` localized ' to just a small area of theregion examined. With enough passages, the recombinantsdisappeared altogether. These results indicated that `recombinationhotspots ' can actually be the result of natural selectionon a pool of random recombination crossover junctions, asopposed to elevated recombination rates in particular regions.Crucially, they also suggested that recombination may bemore common than often assumed, but may go undetectedbecause of the action of strong purifying selection which willremove new, deleterious combinations of mutations. In light ofthese studies it is clear that what is meant by ` recombinationfrequency' ± a term usually used without specified units ±depends critically on whether we are assessing recombinationevents before or after selection has acted. A virus which oftenproduces hybrid RNAs under laboratory conditions may veryrarely ± or even never ± be found to recombine in nature. Thisdifference is analogous to the important distinction betweenthe rate of mutation and the rate of substitution.Negative selection against non-functional hybrids or thosewith decreased fitness may impose the strongest constraints ofall on the appearance of recombinants. In viruses for which theevolutionary costs of recombination outweigh the benefi ts,though they may be mechanistically capable of geneticexchange, strong selection will guarantee the elimination ofhybrid genomes.

ConclusionDetermining the constraints that operate on recombinationoffers a promising path to a fuller understanding of itsimportance in the evolution of RNA viruses. However, outlinesCFEA of the big picture are already clearly visible. It seems certainthat genetic exchange plays a key role in several virus groupsand that it has shaped a good deal of the diversity ± bothancient and recent ± that exists in them. Thus, evolutionaryknowledge about recombination impacts on many aspects ofthe study of RNA viruses, from the broadest investigations ofvirus taxonomy, to the finest details of molecular epidemiologyand vaccine design. A ¯ood of viral gene sequence data and theavailability of new and powerful phylogenetic methods ismaking the detection and characterization of recombinationever easier, and the list of viruses showing recombinationcontinues to grow. The evidence for recombination, not onlybetween closely related viruses but also among distantlyrelated viruses, positive-sense and negative-sense viruses, DIRNAs and viruses, satellite RNAs and viruses, and even withhost RNAs, suggests that almost any genetic material can begrist for the polymerase's mill. Of all the tricks up the viralevolutionary sleeve, surely recombination is one of the mostdeft.